Mustard Chlorambucil Research Articles

Aromatic Nitrogen Mustard-Based Autofluorescent Amphiphilic Brush Copolymer as pH-Responsive Drug Delivery Vehicle.

Delivery of clinically approved nonfluorescent drugs is facing challenges because it is difficult to monitor the intracellular drug delivery without incorporating any integrated fluorescence moiety into the drug carrier. The present investigation reports the synthesis of a pH-responsive autofluorescent polymeric nanoscaffold for the administration of nonfluorescent aromatic nitrogen mustard chlorambucil (CBL) drug into the cancer cells. Copolymerization of poly(ethylene glycol) (PEG) appended styrene and CBL conjugated N-substituted maleimide monomers enables the formation of well-defined luminescent alternating copolymer. These amphiphilic brush copolymers self-organized in aqueous medium into 25-68 nm nanoparticles, where the CBL drug is enclosed into the core of the self-assembled nanoparticles. In vitro studies revealed ∼70% drug was retained under physiological conditions at pH 7.4 and 37 °C. At endolysosomal pH 5.0, 90% of the CBL was released by the pH-induced cleavage of the aliphatic ester linkages connecting CBL to the maleimide unit. Although the nascent nanoparticle (without drug conjugation) is nontoxic, the drug conjugated nanoparticle showed higher toxicity and superior cell killing capability in cervical cancer (HeLa) cells rather than in normal cells. Interestingly, the copolymer without any conventional chromophore exhibited photoluminescence under UV light irradiation due to the presence of "through-space" π-π interaction between the C═O group of maleimide unit and the adjacent benzene ring of the styrenic monomer. This property helped us intracellular tracking of CBL conjugated autofluorescent nanocarriers through fluorescence microscope imaging. Finally, the 4-(4-nitrobenzyl)pyridine (NBP) colorimetric assay was executed to examine the ability of CBL-based polymeric nanomaterials toward alkylation of DNA.

A novel delocalized lipophilic cation-chlorambucil conjugate inhibits P-glycoprotein in HepG2/ADM cells

Multidrug resistance (MDR) limits the application of a large number of cancer-fighting agents in clinical therapy. One reason is that P-glycoprotein (Pgp) efflux pumps are usually overexpressed and lead to drug efflux in the cancer cells, which limits the viability of many chemotherapeutics. Current available inhibitors which block the Pgp pump efflux are usually not widely used in clinical practice, because they change other drug pharmacokinetic profiles or increase side effects. Here, through covalent linkage of cancer-targeting delocalized lipophilic cation FF and DNA-damaging drug nitrogen mustard chlorambucil (CLB), we rationally designed and synthesized a tumor-targeting anticancer agent FFCLB. And we found and proved that the FFCLB was capable of reducing the outflow of Pgp substrates efficiently. This conjugate selectively improves adriamycin uptake and toxicity through reducing MDR1 mRNA and Pgp protein expression. Based on molecular targeted strategy, this study can facilitate the discovery of superior MDR reducing agents to provide a more effective and safer way of resensitizing MDR.

Reprogramming the Mechanism of Action of Chlorambucil by Coupling to a G-Quadruplex Ligand

The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.

A Selective Mitochondrial-Targeted Chlorambucil with Remarkable Cytotoxicity in Breast and Pancreatic Cancers

Nitrogen mustards, widely used as chemotherapeutics, have limited safety and efficacy. Mitochondria lack a functional nucleotide excision repair mechanism to repair DNA adducts and are sensitive to alkylating agents. Importantly, cancer cells have higher intrinsic mitochondrial membrane potential (Δψmt) than normal cells. Therefore, selectively targeting nitrogen mustards to cancer cell mitochondria based on Δψmt could overcome those limitations. Herein, we describe the design, synthesis, and evaluation of Mito-Chlor, a triphenylphosphonium derivative of the nitrogen mustard chlorambucil. We show that Mito-Chlor localizes to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death, resulting in a 80-fold enhancement of cell kill in a panel of breast and pancreatic cancer cell lines that are insensitive to the parent drug. Significantly, Mito-Chlor delayed tumor progression in a mouse xenograft model of human pancreatic cancer. This is a first example of repurposing chlorambucil, a drug not used in breast and pancreatic cancer treatment, as a novel drug candidate for these diseases.

The cap’n’collar transcription factor Nrf2 mediates both intrinsic resistance to environmental stressors and an adaptive response elicited by chemopreventive agents that determines susceptibility to electrophilic xenobiotics

Transcription factor Nrf2 regulates genes encoding drug-metabolising enzymes and drug transporters, as well as enzymes involved in the glutathione, thioredoxin and peroxiredoxin antioxidant pathways. Using mouse embryonic fibroblast (MEF) cells from Nrf2 +/+ and Nrf2 −/− mice, in conjunction with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, we have shown that loss of Nrf2 diminishes the intrinsic resistance of mutant fibroblasts towards isothiocyanates (i.e. sulforaphane), epoxides (i.e. (2S,3S)-(−)-3-phenylglycidol, ethyl 3-phenylglycidate and styrene-7,8-epoxide), peroxides, hydroquinones and quinones (i.e. tert-butylhydroperoxide, tert-butylhydroquinone and 2,3-dimethoxynaphthoquinone), NaAsO 2, and various mutagens, including β-propiolactone, cisplatin, mechlorethamine and methyl methanesulfonate to ∼50% of that observed in equivalent wild-type cells. Exposure of Nrf2 +/+ fibroblasts, but not Nrf2 −/− fibroblasts, to a non-toxic dose (3 μmol/l) of the chemopreventive agent sulforaphane (Sul) stimulated an adaptive response that, 18 h after first being subjected to the isothiocyanate, caused an induction of between 2- and 10-fold in the levels of mRNA for glutamate–cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, glutathione S-transferases and NAD(P)H:quinone oxidoreductase-1 (Nqo1); this was accompanied by an increase in total glutathione of between 1.5- and 1.9-fold. Pre-treatment of Nrf2 +/+ MEF cells with 3 μM Sul for 18 h prior to challenge with xenobiotics, conferred between 2.0- and 4.0-fold protection against isothiocyanates, reactive carbonyls, peroxides, quinones, NaAsO 2, and the anticancer nitrogen mustard chlorambucil, but pre-treatment with 3 μM Sul produced no such increased tolerance in Nrf2 −/− MEF cells. The inducible resistance towards acrolein, cumene hydroperoxide and chlorambucil, produced by pre-treating wild-type fibroblasts with 3 μM Sul, was dependent on glutathione because simultaneous pre-treatment with 5 μmol/l buthionine sulfoximine abolished the increased tolerance of these xenobiotics. However, inducible resistance towards menadione that occurred upon pre-treatment with 3 μM Sul was independent of glutathione and may be due to upregulation of Nqo1. Thus Nrf2 controls cellular resistance against electrophiles.

Cytokinetics and mechanism of action of AKO4: a novel nitrogen mustard targeted to bcr-abl

The “combi-targeting” concept seeks to design molecules to not only block tyrosine kinase (TK) activity but also to induce DNA damage. Here we design AK04, a molecule that combines the pharmacophore chlorambucil with that of STI-571 (Gleevec™). The results showed that although a less potent abl TK inhibitor than STI571, AK04 was capable of significantly blocking bcr-abl phosphorylation not only in a purified abl assay but also in the bcr-abl+ K562 cells. In contrast to STI571 and like chlorambucil, it induced a dose-dependent increase in DNA damage in these cells. More importantly, AK04 was 12–32-fold more potent than chlorambucil in all bcr-abl+ cells of our cell panel. In the isogenic human megakaryocytic Mo7e and Mo7/bcr-abl cells, AK04 selectively killed the bcr-abl transfectants. Flow cytometry revealed that despite being a five-fold less potent inhibitor of bcr-abl than STI-571, it induced a significant dose-dependent increase in levels of cell death by apoptosis in KU812 cells 24 h post-treatment. Under these conditions, chlorambucil did not induce any significant level of apoptosis. These results suggest that AK04 is a nitrogen mustard with binary bcr-abl/DNA targeting effects, a property that may account for its superior potency when compared with the classical mustard chlorambucil.

Induction of apoptosis by the ADEPT agent ZD2767: comparison with the classical nitrogen mustard chlorambucil and a monofunctional ZD2767 analogue.

ZD2767P is a phenol mustard glutamate prodrug which is currently being developed for Antibody Directed Enzyme Prodrug Therapy (ADEPT). In ZD2767 ADEPT an active bi-functional alkylating drug, ZD2767D (4-[N,N-bis(2-iodoethyl)amino]phenol), is generated following cleavage of ZD2767P by the bacterial enzyme carboxypeptidase G2 (CPG2) which is targeted to the tumour by conjugation to the F(ab′)2 fragment of the anti-CEA antibody A5B7. The aim of the studies described here was to identify the mode of cell death induced by ZD2767P + CPG2 in comparison to the established nitrogen mustard chlorambucil. The contribution of bifunctional and monofunctional ZD2767 DNA lesions to cell death induction was investigated using a monofunctional ZD2767D analogue. Apoptosis in LoVo tumour cells was studied by three different methods (nuclear morphology, annexin V staining and TUNEL). Levels of apoptosis detected using the three assays were similar, and each drug treatment produced apoptosis at levels above those in control cells at concentrations which resulted in tumour cell growth inhibition. The bi-functional compounds, ZD2767P + CPG2 and chlorambucil, induced apoptosis in a concentration and time dependent manner, with equitoxic concentrations producing equivalent levels of apoptosis. In contrast, the mono-functional ZD2767D analogue at 100 μM resulted in the maximal level of apoptosis at 25 h with no further increase over the following 72 h. These studies have demonstrated that apoptosis is the mechanism of cell death induced by the ZD2767 ADEPT system, and that levels of apoptosis produced by ZD2767 are similar to those observed at equitoxic concentrations of the classical nitrogen mustard chlorambucil. The mono-functional ZD2767 analogue also induced apoptosis, but with a different time course and characteristics. In conjunction with previous data, these studies have shown that the potent activity of ZD2767 can be attributed to the ability of the compound to induce bifunctional DNA lesions and engage apoptosis. © 2001 Cancer Research Campaign http://www.bjcancer.com

Genetic damage by bifunctional agents in repair-active pre-meiotic stages of Drosophila males

Most of our understanding of germline mutagenesis in Drosophila is based on the DNA repair-inactive, haploid post-meiotic stages. The diploid, repair-active pre-meiotic stages are more relevant to the situation encountered in somatic cells. DNA mono-adducts induced by agents like methyl methanesulphonate (MMS) and ethylene oxide (EO) are well repaired in the pre-meiotic cell stages, and these agents show therefore, no or considerable lower mutagenic activity in these stages. In contrast, in this study the two bifunctional nitrogen mustards chlorambucil (CAB) and mechlorethamine (MEC) show significantly elevated mutant frequencies of both post- and pre-meiotic germ cells. Results were similar for the X-chromosomal and the autosomal (2nd) recessive lethal (RL) test. CAB and MEC were also active in stem cells, but in comparison with post-stem cell stages they seem to be better protected. The germ cell specific response in post- and pre-meiotic cell stages was for both nitrogen mustards comparable to mutagenic activity patterns observed in the specific locus test in the mouse. It was reported that for diepoxybutane (DEB), another cross-linking agent, the ratio of the RL frequency for the 2nd- and the X-chromosome was increased from 2.1 for post-meiotic stages to 9.5 for pre-meiotic stages. In own experiments aiming to confirm this observation, a high ratio was indeed found. The induction of large deletions by DEB could be the reason for this difference, since such lesions might include both a sex-linked lethal and a vital gene required for the development of spermatocytes into mature sperm. Similar differences were expected for CAB and MEC since they are also inducers of large deletions. But unexpectedly, no differences in 2nd/X RL ratio between post- and pre-meiotic cell stages were found for the nitrogen mustards. Possible causes such as distinct proportions of multi-locus deletions (MLDs), mitotic recombination and the formation of persistent lesions, are discussed.

The mutagenic spectrum of acridine-linked aniline nitrogen mustards in AS52 cells: implications of DNA targeting with high selectivity for adenine or guanine bases

The mutational spectra generated in AS52 cells at the gpt gene locus by aniline mustards were studied by the isolation of resistant clones and sequencing of the altered gene. A set of four aniline mustards (both mono- and bifunctional) linked to a DNA-affinic 9-aminoacridine (9-AA) carrier was used, together with the untargeted mustards chlorambucil (CHL) and its half-mustard, and the DNA binding carrier, 9-AA. Both 9-AA and CHL were weak cytotoxins, with the DNA-targeted mustards being markedly (10–40-fold) more dose potent, and the bifunctional ones somewhat more toxic than the monofunctional ones. 9-AA produced a different spectrum of mutations to the spontaneous background, with more minor addition events and less base pair substitutions, and showing for the first time that frameshift events so characteristic of 9-AA in bacteria or bacteriophage also occur in mammalian cells. The mutational spectra of the DNA-targeted mustards were quite different both from this and from the lesions caused by the untargeted mustards, which cause largely transition mutations at AT sites (despite a clear preference for formation of N 7-guanine adducts). There were very few transition mutations, suggesting that the initial O 6-alkylguanine/ O 4-alkylthymine lesions considered to give rise to these are relatively rare. There was also a lower incidence of complete deletions, usually attributed to DNA cross-links. For the short chain length targeted mustards, which form initial stable adducts largely (95%) at guanine N 7 sites, base pair substitution mutations, predominantly transversions, involved AT and GC base pairs equally. In contrast, the longer chain length targeted mustards, which form >90% of initial adducts at adenine N 1 sites, generated also formed transversion mutations, but these overwhelmingly (24/27) involved AT base pairs.

Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents

There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites, in addition to sites characteristic of untargeted mustards.

Comparative mutational spectra of the nitrogen mustard chlorambucil and its half-mustard analogue in Chinese hamster AS52 cells

Nitrogen mustards play an important role in current cancer chemotherapy. The most effective antitumour agents are those carrying two alkylating functions, probably through their ability to form interstrand cross-links in DNA. Such lesions appear to create more of a block in DNA replication and are more difficult to repair than are most monoadducts. Although there were early reports that monofunctional drugs were more mutagenic than the bifunctional drugs, this has not been formally proved using structurally related drugs in a mutagenicity assay capable of detecting a range of different events. We have studied both the mutagenic potency and spectrum of events caused by treatment with the clinical agent, chlorambucil, compared with its half-mustard analogue, in Chinese hamster ovary (CHO)-AS52 cells. Although both drugs caused comparable increases in mutation frequency at doses killing 90% of cells (from around 9×10 −6 to around 9×10 −5 mutant cells), the nature of events differed significantly between the drugs. By far the majority of mutations caused by the half-mustard were transversion mutations, and almost all of these could be interpreted in relation to the DNA adducts that are known to be formed. In contrast, the majority of chlorambucil-induced mutations were major deletions, and point mutations were only identified from a few clones. Parallel micronucleus assays verified that chlorambucil has a stronger ability to break chromosomes than the half-mustard. These two drugs are thought to form similar monoadducts, but only the full mustard can form interstrand cross-links. The data suggest that DNA cross-links, although only a minor fraction of the total lesions, dominate the mutagenic spectrum and lead to gross changes at the chromosome level that can not be readily associated with individual lesions produced by the drug.

DNA alkylation sites of nitrogen mustards conjugated to polyamines and their implications for polyamine–DNA interactions

Polyamines conjugated to the nitrogen mustard chlorambucil increase the efficiency of DNA alkylation at N7 of guanine by factors in the range 103 to 104; the sequence selectivity of this alkylation (the alkylation ‘finger-print’) is largely unchanged, which is consistent with flexible, electrostatic binding and incompatible with tight, sequence-specific binding of the polyamine moiety.

RecA-catalyzed, sequence-specific alkylation of DNA by cross-linking oligonucleotides. Effects of length and nonhomologous base substitutions.

Oligodeoxyribonucleotides (ODNs) bearing the reactive nitrogen mustard chlorambucil have been used as sequence-directed affinity labeling reagents to investigate the length and homology requirements for RecA-catalyzed alkylation of double-stranded DNA. The cross-linkage reaction, which takes place at the N-7 position of a targeted complementary strand guanine following strand exchange, was highly sequence specific with both a 272 bp DNA fragment and a linearized plasmid. Alkylation required the ODN to be at least 26 nucleotides long and to possess homology to the target in the vicinity of the modification site. The extent of alkylation was improved by using longer ODNs, with a 50-mer giving over 50% reaction. Mismatches inhibited alkylation when they perturbed the structure of the strand exchange product near the targeted guanine. Longer heterology also inhibited alkylation when it prevented strand exchange. Our inability to detect cross-linkage in stable synaptic complexes unable to undergo complete strand exchange is best explained by a model for homologous alignment in which the presynaptic filament approaches from the minor groove of the duplex. Since the N-7 position of guanine is in the major groove, it is inaccessible to the tethered chlorambucil group of the ODN during the search for homology. The reaction specificity of chlorambucil-bearing ODNs suggests that they may have general use as recombinase-mediated DNA targeting agents.

Synthesis, DNA interactions and biological activity of DNA minor groove targeted polybenzamide-linked nitrogen mustards

A series of polybenzamide DNA minor groove binding ligands bearing either one or two monofunctional mustards have been synthesised, and their cytotoxicities and interactions with DNA have been studied. Analogues with two alkylating functions (e.g. compounds 7 and 14) are the most cytotoxic, with 7 being 1000-fold more potent than the clinical - mustard chlorambucil against P388 leukemia in culture, as well as being more potent in vivo. Monofunctional analogues were also significantly more cytotoxic than chlorambucil, despite bearing much less reactive mustard species. These results support the concept that targeting nitrogen mustard alkylating agents to DNA by attachment to DNA-affinic carriers can greatly enhance cytotoxicity due to alkylation, and that even for such DNA-targeted mustards, crosslinking is a more toxic event than monoalkylation. Close analogues of 7 differing only in their radius of curvature, appear to alkylate and crosslink DNA in similar fashion, yet have widely differing cytotoxicities. The most cytotoxic compound (7) possesses a geometry most complementary to that of duplex DNA, suggesting that the most toxic lesions are those which result in least DNA distortion, thus being less easily recognised by DNA repair systems.

Synthesis and cytotoxic activity of new alkyl[3-(2-chloroethyl)ureido]benzene derivatives

Several alkyl[3-(2-chloroethyl)ureido] (CEU) benzene derivatives were prepared as potential anticancer agents. These new compounds were readily prepared in good yields by addition of anilines to 2-chloroethylisocyanate. Their cytotoxic activity was evaluated on human breast cancer (MDA-MB-231), human colon adenocarcinoma (LoVo) and mouse lymphocytic leukemia (P388D 1) tumor cell lines. Several new CEUs were significantly more cytotoxic than the nitrogen mustard chlorambucil. The biological activity of these aromatic urea derivatives seems to be related to the nature and position of the alkyl substituents on the aromatic ring. Substitution by branched alkyl groups on position 4 of the aromatic ring led to cytotoxic molecules which are up to 5 times more potent than the standard chlorambucil.

Chlorambucil-induced cell death in embryonic mouse limb buds

A high frequency of congenital limb defects after maternal treatment with the nitrogen mustard chlorambucil suggested that cytotoxic effects could be analyzed as correlates of teratogenic action of this alkylating agent. Limb buds from ICR DUB mouse embryos were exposed in vivo or in vitro to teratogenic doses of chlorambucil. At 0.5- to 72-hr intervals following exposure, the limbs were processed for observation with the light and electron microscopes. Light microscopy of limb buds exposed to the drug in vivo or in vitro showed evidence of cellular involvement within 4 hr following treatment. The effects were manifested in the form of cytoplasmic inclusions which increased in number as the time following exposure increased, and, in vivo, reached a maximum at 24 hr. Increased doses of the drug also produced increased numbers of affected cells. In limb buds exposed to chlorambucil in vitro, the number of affected cells reached a plateau at 48 hr after treatment and decreased significantly by 72 hr, at which time exposure to a second dose of the drug had little or no effect. Affected cells were restricted almost exclusively to regions of undifferentiated mesenchymal cells. Electron microscopic examination of the cytoplasm of affected cells demonstrated the presence of membrane-bound vacuoles containing cellular organelles in various stages of degeneration. The number and size of these vacuoles increased with time following exposure until finally the cells fragmented. Nuclear material was not involved until fragmentation of the cells occurred, at which time the nucleus became pyknotic. Following cellular fragmentation and destruction, macrophages were observed in association with the cellular debris. Acid phosphatase was demonstrated within the vacuoles (by the presence of lead phosphate precipitates) with the electron microscope after incubating limb bud tissue in a buffered solution of β-glycerophosphate/lead nitrate. The amount of demonstrable enzyme was found to increase as the degree of breakdown within vacuoles increased. These results and the histological information provided evidence consistent with a process of autophagocytosis.

Augmentation of cytotoxic drug action by antibodies directed at cell surface.

THE concept of attaching cytotoxic agents to tumour-specific antibodies in the treatment of cancer is therapeutically attractive. In this way the specificity of the agents would be increased and their systemic toxicity reduced. This immunochemotherapeutic approach has been successfully applied in experimental systems when the cytotoxic agent has been covalently bound to the antibody1–3. Similar success has been reported with the nitrogen mustard chlorambucil, merely physically adsorbed on to the globulin fraction from tumour-specific antiserum without the actual formation of a covalent bond4. It is surprising that antibody could act as a carrier for chlorambucil under these conditions, as has been proposed5, as the two molecules would be expected to dissociate in the in vivo environment. It is, therefore, probable that the increased cytotoxicity of chlorambucil in the presence of antibody is attributable to some other mechanism. The experiments described here are an attempt to elucidate this problem further.